An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) betaglucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.     

Uptake of fluorescent fatty acids by erythroleukemia cells. Effect of differentiation

The uptake of a fluorescent derivative of a fatty acid (FDFA), 12-(1-pyrene) dodecanoic acid (P12) by murine erythroleukemia (MEL) cells was studied. Because of the intense fluorescence of the pyrene ring, the association of P12 with intact cells could be analysed using a fluorescence microscope or a fluorescence-activated cell sorter (FACS), and the incorporation of P12 into cellular lipids could be quantified, following their extraction in a spectrofluorimeter. These procedures indicated that P12 uptake and intracellular utilization are reduced, following induction of erythrodifferentiation by dimethylsulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA). The differences in the fluorescence observed following exposure to P12 permitted us to separate a mixture of differentiated and undifferentiated cells into two distinct cell subpopulations; the high fluorescence population consisted mainly of undifferentiated cells, and the low one of differentiated cells. The results of this study suggest that fluorescent fatty acids are useful for distinguishing between and sorting cells at different stages of differentiation.     

Class similarity and viewpoint invariance in the recognition of 3D objects

 In human vision, the processes and the representations involved in identifying specific individuals are frequently assumed to be different from those used for basic level classification, because classification is largely viewpoint-invariant, but identification is not. This assumption was tested in psychophysical experiments, in which objective similarity between stimuli (and, consequently, the level of their distinction) varied in a controlled fashion. Subjects were trained to discriminate between two classes of computer-generated three-dimensional objects, one resembling monkeys and the other, dogs. Both classes were defined by the same set of 56 parameters, which encoded sizes, shapes, and placement of the limbs, ears, snout, etc. Interpolation between parameter vectors of the class prototypes yielded shapes that changed smoothly between monkey and dog. Within-class variation was induced in each trial by randomly perturbing all the parameters. After the subjects reached 90% correct performance on a fixed canonical view of each object, discrimination performance was tested for novel views that differed by up to 60° from the training view. In experiment 1 (in which the distribution of parameters in each class was unimodal) and in experiment 2 (bimodal classes), the stimuli differed only parametrically and consisted of the same geons (parts), yet were recognized virtually independently of viewpoint in the low-similarity condition. In experiment 3, the prototypes differed in their arrangement of geons, yet the subjects’ performance depended significantly on viewpoint in the high-similarity condition. In all three experiments, higher interstimulus similarity was associated with an increase in the mean error rate and, for misorientation of up to 45°, with an increase in the degree of viewpoint dependence. These results suggest that a geon-level difference between stimuli is neither strictly necessary nor always sufficient for viewpoint-invariant performance. Thus, basic and subordinate-level processes in visual recognition may be more closely related than previously thought. Received: 15 November 1993/Accepted in revised form: 14 July 1994     

Molecular Exploration of the First-Century Tomb of the Shroud in Akeldama,Jerusalem

The Tomb of the Shroud is a first-century C.E. tomb discovered in Akeldama, Jerusalem, Israel that had been illegally entered and looted. The investigation of this tomb by an interdisciplinary team of researchers began in 2000. More than twenty stone ossuaries for collecting human bones were found, along with textiles from a burial shroud, hair and skeletal remains. The research presented here focuses on genetic analysis of the bioarchaeological remains from the tomb using mitochondrial DNA to examine familial relationships of the individuals within the tomb and molecular screening for the presence of disease. There are three mitochondrial haplotypes shared between a number of the remains analyzed suggesting a possible family tomb. There were two pathogens genetically detected within the collection of osteological samples, these were Mycobacterium tuberculosis and Mycobacterium leprae. The Tomb of the Shroud is one of very few examples of a preserved shrouded human burial and the only example of a plaster sealed loculus with remains genetically confirmed to have belonged to a shrouded male individual that suffered from tuberculosis and leprosy dating to the first-century C.E. This is the earliest case of leprosy with a confirmed date in which M. leprae DNA was detected.     

A compatible interaction of Alternaria brassicicola with Arabidopsis thaliana ecotype DiG: evidence for a specific transcriptional signature

Background  

The interaction of Arabidopsis with Alternaria brassicicola provides a model for disease caused by necrotrophs, but a drawback has been the lack of a compatible pathosystem. Infection of most ecotypes, including the widely-studied line Col-0, with this pathogen generally leads to a lesion that does not expand beyond the inoculated area. This study examines an ecotype, Dijon G (DiG), which is considered sensitive to A. brassicicola.     

Network Events on Multiple Space and Time Scales in Cultured Neural Networks and in a Stochastic Rate Model

Cortical networks, in-vitro as well as in-vivo, can spontaneously generate a variety of collective dynamical events such as network spikes, UP and DOWN states, global oscillations, and avalanches. Though each of them has been variously recognized in previous works as expression of the excitability of the cortical tissue and the associated nonlinear dynamics, a unified picture of the determinant factors (dynamical and architectural) is desirable and not yet available. Progress has also been partially hindered by the use of a variety of statistical measures to define the network events of interest. We propose here a common probabilistic definition of network events that, applied to the firing activity of cultured neural networks, highlights the co-occurrence of network spikes, power-law distributed avalanches, and exponentially distributed ‘quasi-orbits’, which offer a third type of collective behavior. A rate model, including synaptic excitation and inhibition with no imposed topology, synaptic short-term depression, and finite-size noise, accounts for all these different, coexisting phenomena. We find that their emergence is largely regulated by the proximity to an oscillatory instability of the dynamics, where the non-linear excitable behavior leads to a self-amplification of activity fluctuations over a wide range of scales in space and time. In this sense, the cultured network dynamics is compatible with an excitation-inhibition balance corresponding to a slightly sub-critical regime. Finally, we propose and test a method to infer the characteristic time of the fatigue process, from the observed time course of the network’s firing rate. Unlike the model, possessing a single fatigue mechanism, the cultured network appears to show multiple time scales, signalling the possible coexistence of different fatigue mechanisms.